1. Field of the Invention
The invention relates to a method of finding, recording and optionally evaluating object structures, preferably of fluorescent object structures such as gene spots, on slides. A microscope with a CCD camera, a scanning microscope, or a confocal laser scanning microscope can be used for recording.
2. Related Prior Art
In a very wide range of applications, for example in the context of an automatic production process, individualxe2x80x94arbitraryxe2x80x94object structures are localized. It is often necessary for object structures to be found again and to be classified with a predefined reliability.
In conventional techniques in which so-called gene scanners have hitherto been used, slides or fluorescent spots arranged on them and having a diameter on the order of 5 xcexcm and 100 xcexcm are imaged. This may be a conventional microscopic image which is recorded, for example, in conjunction with a CCD camera. Likewise, a scanning microscope or a confocal laser scanning microscope (or xe2x80x9cCLSMxe2x80x9d) may be used. In a confocal laser scanning microscope, the detection technology is preferred because the dynamic range which can be detected is generally higher.
In the process of recording using conventional confocal laser scanning microscopes, images are recorded with a very high positional resolution, so that, for object identification, for example, computer-aided segmentation is subsequently necessary. This is accompanied by a very high expenditure in terms of time and requires a considerable computer capacity. With regard to the detectability, the requirements on the recording and evaluation methods are normally around one molecule per square micrometer (xcexcm2) and about one percent (1%).
The gene scanners which are known from practice, as well as the methods which are applied with them are extremely complicated, on account of (1) the data recording which is necessary there with anxe2x80x94unnecessarilyxe2x80x94high positional resolution, and (2) the subsequently necessary segmentation, with a high computer outlay on account of the necessary capacity, and which necessitates a considerable outlay on apparatus. In addition, the segmentation of the individual fluorescent objects, which is to be carried out with a computer, is often subject to error, so that the required accuracy of about 1% is not always achieved. To this extent, the methods which have hitherto been known from practice are inadequate.
In view of the foregoing, an object of the present invention is to provide a method of finding, recording and optionally evaluating object structures, especially on slides, preferably of fluorescent object structures such as gene spots, for example, in which method rapid and adequately reliable detection of the object structures is possible. The detection of the object structures must be efficient, it being intended for a specific evaluation method to permit the accurate, rapid and simultaneously reliable identification and localization of the object structures. In the case of detecting fluorescent gene spots, care must be taken that the saturation of the fluorochromes is limiting for the scanning speed. In any case, account should be taken of the fact that fluorescent objects are only able to emit a limited fluorescence intensity.
The preceding object can be achieved by the features described herein. According to one embodiment, a method of acquiring object structures of an object comprises projecting an illumination pattern onto an object plane where the object is located and detecting an object structure of the object with a microscope having a light source and a detector.
In another embodiment of the present invention, a method of finding, recording and optionally evaluating object structures comprises recording image data using an illumination pattern projected into the object plane. For example, fluorescent object structures on slidesxe2x80x94such as gene spots can be recorded using a microscope with a CCD camera, a scanning microscope or a confocal laser scanning microscope, with the image data being recorded using an illumination pattern projected into the object plane.
In yet another embodiment of the present invention, an optical detection device for detecting, recording and evaluating fluorescent object structures of an object includes a confocal laser scanning microscope having a light source and a detector, where the object is located in an object plane of said microscope. The device also includes a first illumination mask for projecting an illumination pattern onto the object, where the first illumination mask is disposed along an illumination beam path of said microscope and preferably (not necessarily) arranged symmetrically to an optical axis of said microscope. The projected illumination pattern on the object generates a back reflection or fluorescence distribution from the object structures. The device includes a first detection mask disposed along a detection beam path of the microscope, where the back reflection or fluorescence distribution from the object structures is detected via the first detection mask. An image data storage unit can be used to store extracted object information from the object structures.
Further features of the invention form the subject matter of the claims and will be explained in more detail, in conjunction with further advantages of the invention, with reference to an exemplary embodiment.